For many years, antibiotics have been used to combat bacterial infection, most commonly in the hospital setting, and have thus become an integral part of our lives. Since the discovery of penicillin by Dr. Alexander Fleming in 1928, many generations of β-lactam antibiotics which target the cell wall synthesis pathway have been introduced. However, since the introduction of β-lactam antibiotics, over the years bacteria have developed significant resistance. The earliest resistance of S. aureus and other bacteria comes from a family of proteins called penicillinases, which have the capability of binding penicillins, hydrolyzing the β-lactam ring, thus rendering the inactivity of the antibiotics.
One such bacteria, Staphylococcus aureus, commonly found in hospital settings on the skin and inside the noses of humans and animals, has quickly evolved into a “super-bug”. Previously, methicillin, a semi-synthetic penicillin derivative, was designed to have a rigid β-lactam ring and to disrupt the cell wall synthesis pathway of these bacteria. However, resistance from S. aureus began to appear just one year after Methicillin's introduction, and Methicillin-resistant S. aureus (MRSA) have quickly spread to hospitals worldwide where there is an alarmingly high prevalence of these bacteria. It is thought that this bacterial resistance arises from the acquisition of the mecA gene via horizontal transfer from an unidentified species. The mecA gene encodes a new penicillin-binding protein (PBP2a), which has unusually low β-lactam affinity and remains active to allow cell wall synthesis even at normally lethal antibiotics concentrations. As MRSAs are resistant to other classes of antibiotics, glycopeptides such as vancomycin emerged as the possible last defense against MRSA infections. Unfortunately, but not surprisingly, scientists and clinicians have now recognized the appearance of a vancomycin-resistant MRSA strain (also referred to as Vancomycin-intermediate S. aureus or VISA), first in Japan in 1996, and subsequently in the United States, France, Korea, South Africa and Scotland, and this further emphasizes the urgent need for new and more effective antibiotics which would be harder for bacteria to overcome and develop resistance against
The problems presented by bacteria and other pathogenic agents have become more dangerous since Sep. 11, 2001 and the anthrax scare in the fall of 2001, and bioterrorism has become a serious concern of our nation and the world. In fact, S. aureus was identified by CDC as a level 2 bioterrorism threat, due to the lethal toxicity from the Staphylococcal Enterotoxin B. Bacteria such as S. aureus also affect other industries such as milk production in that the inflammatory infection of S. aureus in cows' mammary glands, Mastitis, causes them to produce poor-quality milk that can not be made into cheese, butter or yogurt. This costs the dairy industry an estimated $1.7 billion in the United States alone.
One avenue for fighting and treating bacterial infection that has been studied over the years has been the use of lysozymes. Lysozymes are enzymes which attack bacteria by cleaving their oligosaccharide backbone of the cell walls, a mechanism different from the β-lactams, and many bacteria have been observed to have difficulties developing resistance for it. One particular type of enzyme, the Lysozyme from fungi of the species Chalaropsis, which is known as Chalaropsis lysozyme, or Lysozyme Ch, and which is a N,O-diacetylmuramidase, has the ability to kill S. aureus and has previously been thought of as a good agent to be used in antibacterial agents. It can also be developed as an early detection method of staphylococcal contaminations or infections, and this family of Lysozymes has been shown to significantly reduce the total number of both Gram-positive and Gram-negative bacteria when applied in meat products, including Clostricium Botulinum, identified by CDC as the number two bioterrorism threat, second to smallpox. It is thought that the action of Lysozymes cleave the oligosaccharide backbone of the bacterial cell wall, resulting a weakened cell wall and eventual cell lysis and death. Lysozyme Ch, produced extracellularly by the fungus Chalaropsis species, is unusual among Lysozymes by having both β-1,4-N-acetylmuramidase and β-1,4-N,6-O-diacetylmuramidase activities. The later activity accounts for its ability to kill S. aureus whose cell walls contain 6-O acetylated peptidoglycans such as N,6-O-diacetylmuramic acid, and recent studies with an enzyme of bacteriophage γ, PlyG, being highly effective for killing B. anthracis, demonstrate the achievability of anti-bacterial Lysozymes. Moreover, the bacteria did not develop a resistance even after being treated with chemicals which normally increase the percentage of mutants resistant to standard antibiotics.
The present inventors have now determined that although the sequence of Lysozyme was published, this actual sequence is wrong. Accordingly, if one tried to generate recombinant lysozyme using the previously published sequence, it would not have the activity of the correct sequence. It is thus a highly desirable object to obtain the correct sequence of Lysozyme Ch so as to be able to use this protein as a solution to the ever growing problem of bacterial infections such as MRSA and VISA. It thus remains a highly desirable object to be able to provide a Lysozyme Ch of maximal antibacterial activity so as to develop more effective methods of treating and preventing the spread of bacteria which will be of great importance in hospitals, veterinary medicine, and in the fight against bioterrorism.